1. Field of Invention
The present invention relates to a mass spectrometer in general and in particular to a tandem mass spectrometer that combines two time-of-flight mass spectrometers.
2. Description of Related Art
Mass spectrometers are instruments that are used to determine the chemical composition of substances and the structures of molecules. In general they consist of an ion source where neutral molecules are ionized, a mass analyzer where ions are separated according to their mass/charge ratio, and a detector. Mass analyzers come in a variety of types, including magnetic field (B) instruments, combined electrical and magnetic field or double-focusing instruments (EB or BE), quadrupole electric field (Q) instruments, and time-of-flight (TOF) instruments. In addition, two or more analyzers may be combined in a single instrument to produce tandem (MS/MS) mass spectrometers. These include triple analyzers (EBE), four sector mass spectrometers (EBEB or BEEB), triple quadrupoles (QqQ) and hybrids (such as the EBqQ).
In tandem mass spectrometers, the first mass analyzer is generally used to select a precursor ion from among the ions normally observed in a mass spectrum. Fragmentation is then induced in a region located between the mass analyzers, and the second mass analyzer is used to provide a mass spectrum of the product ions. Tandem mass spectrometers may be utilized for ion structure studies by establishing the relationship between a series of molecular and fragment precursor ions and their products. Alternatively, they are now commonly used to determine the structures of biological molecules in complex mixtures that are not completely fractionated by chromatographic methods. These may include mixtures of, for example, peptides, glycopeptides or glycolipids. In the case of peptides, fragmentation produces information on the amino acid sequence.
One type of mass spectrometers is time-of-flight (TOF) mass spectrometers. The simplest version of a time-of-flight mass spectrometer, illustrated in FIG. 1 (Cotter, Robert J., Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research, American Chemical Society, Washington, D.C., 1997), the entire contents of which is hereby incorporated by reference, consists of a short source region 10, a longer field-free drift region 12 and a detector 14. Ions are formed and accelerated to their final kinetic energies in the short source region 10 by an electric field defined by voltages on a backing plate 16 and drawout grid 18. Other grids or lenses 17 may be added to the source region to enhance extraction and to improve the mass resolution. The longer field-free drift region 12 is bounded by drawout grid 18 and an exit grid 20.
In the most common configuration, the drawout grid 18 and exit grid 20 (and therefore the entire drift length) are at ground potential, the voltage on the backing plate 16 is V, and the ions are accelerated in the source region to an energy: mv2/2=z eV, where m is the mass of the ion, v is its velocity, e is the charge on an electron, and z is the charge number of the ion. The ions then pass through the drift region 12 and their (approximate) flight time(s) is given by the formula:t=[(m/z)/2 eV]1/2D  (I)which shows a square root dependence upon mass. Typically, the length 1 of source region 10 is of the order of 0.5 cm, while drift length (D) ranges from 15 cm to 8 meters. Accelerating voltages (V) can range from a few hundred volts to 30 kV, and flight times are of the order of 5 to 100 microseconds. Generally, the accelerating voltage is selected to be relatively high in order to minimize the effects on mass resolution arising from initial kinetic energies and to enable the detection of large ions. For example, the accelerating voltage of 20 KV (as illustrated for example in FIG. 1) has been found to be sufficient for detection of masses in excess of 300 kDaltons (kDa).
Mass resolution can be improved by pulsing one or more of the source elements such as the backing plate 16 or the grid 17. Other time-dependent pulses or waveforms may also be applied to the source (Kovtoun, S. V., English, R. D. and Cotter, R. J., Mass Correlated Acceleration in a Reflectron MALDI TOF Mass Spectrometer: An Approach for enhanced Resolution over a Broad Range, J. Amer. Soc. Mass Spectrom. 13 (2002) 135-143).
Mass resolution may also be improved by the addition of a reflectron (Mamyrin, B. A., Karataev, V. I., Shmikk, D. V. Zagulin, V. A. Sov. Phys. JETP 37 (1973) 45). A conventional reflectron is essentially a retarding electrical field which decelerates the ions to zero velocity, and allows them to turn around and return along the same or nearly the same path. Ions with higher kinetic energy (velocity) penetrate the reflectron more deeply than those with lower kinetic energy, and thus have a longer path to the detector. Ions retain their initial kinetic energy distributions as they reach the detector; however, ions of different masses will arrive at different times.
An example of a time-of-flight mass spectrometer utilizing a reflectron is shown schematically in FIG. 2 (same numerals in FIG. 1 and FIG. 2 are used to indicate same elements however positioned differently). The reflectron may be single stage 30 or dual-stage. In both single-stage and dual-stage reflectrons, a stack of electrodes 32 (also called ion lenses), each connected resistively to one another, provide constant retarding field regions that are separated by one grid 34 in the single stage reflectron 30. In the most common case, grids and lenses are constructed using ring electrodes. In the case of grid 34 illustrated in FIG. 2, the ring electrode is covered with a thin wire mesh.
In single-stage reflectrons, a single retarding region is used and approximate ion flight times are given by the formula:t=[(m/z)/2 eV]1/2[L1+L2+4d]  (II)which has the same square-root dependence expressed in Equation (I). The terms, in addition to those expressed in Equation (I), are L1, L2 and d. L1 and L2 are the lengths of the linear drift regions illustrated in FIG. 2, respectively, in the forward and return directions, and d is the average penetration depth. The focusing action can be understood by replacing the denominator in equation (II) with 2 eV+U0, where U0 represents the contribution to the ion velocity from the initial kinetic energy distribution.
While reflectrons were originally intended to improve mass resolution for ions formed in an ion source region, they have more recently been exploited for recording the mass spectra of product ions formed outside the source by metastable decay or by fragmentation induced by collisions with a target gas or surface, by photodissociation or by electron impact. Ions resulting from the fragmentation of molecular ions in the flight path can be observed at times given by the following formula:t=[(m/z)/2 eV]1/2[L1+L2+4(m′/m)d]  (III)where m′ is the mass of the new fragment ion. In the case of peptides, these ions can provide amino acid sequences. The focusing action can be understood by replacing the denominator in equation (III) with 2 eV+U0, where U0 represents the contribution to the ion velocity from the initial kinetic energy distribution. These ions are generally focused by stepping or scanning the reflectron voltage VR or by using non-linear reflectrons, such as the curved-field reflectron described by Cornish and Cotter (Cornish, T. J., Cotter, R. J., Non-linear Field Reflectron, U.S. Pat. No. 5,464,985, the entire content of which is hereby incorporated by reference).
Product ions will appear in normal mass spectra as generally weak and poorly-focused peaks which cannot be easily associated with a given precursor ion. However, it is possible to record the product ion mass spectrum for a single precursor, by selecting ions of a single mass for passage through the first drift region. An example of this approach is described by Schlag et al. (Weinkauf, R.; Walter, K.; Weickhardt, C.; Boesl, U.; Schlag, E. W.: Int. J. Mass Spectrom. Ion Processes Vol. 44A (1989) pp. 1219-25), in which an electrostatic gate is located in the first drift region. The ions passed by the gate are then fragmented by photodissociation using a pulsed UV laser, and the product ions are detected after reflection.
An alternative approach was introduced by LeBeyec and coworkers using a coaxial dual-stage reflectron, and has been developed by Standing et al. (Standing, K. G.; Beavis, R.; Bollbach, G.; Ens. W.; LaFortune, F.; Main, D.; Schueler, B.; Tang, X; Westmore, J. B. Analytical Instrumentation 16(1) (1987) pp. 173-89) using a single-stage reflectron. In this approach, all ions are permitted to enter the reflectron. A detector is also located at the rear of the reflectron and records neutral species resulting from the metastable decay in the first field-free drift length. Because these neutrals appear at time corresponding to the mass of the precursor ion, it is then possible to only register ions in the reflectron detector when a neutral corresponding to the precursor mass is received. The resultant spectrum, known as a correlated reflex spectrum, can only be obtained with methods that employ single ion pulse counting.
A major limitation of the reflectrons designed to date is that focusing of product ions (mass resolution) is not constant over the mass range. Specifically, the selected precursor ion mass is generally the most well focused ion in the product ion mass spectrum, while focusing decreases for product ions with lower mass. This is generally attributed to the fact that lower mass product ions do not penetrate the reflectron to as great a depth as ions whose masses are close to the precursor ion mass. Thus, it has been a common observation that lowering the reflection voltages permits recording of the low mass portion of the spectrum with considerably better focus, while the higher mass ions simply pass through the back end of the reflectron.
For this reason, several investigators have suggested stepping the reflectron voltages to record different regions of the mass spectrum, or scanning the reflectron voltages and reconstructing a focused mass spectrum from a series of transients (Weinkauf, R.; Walter, K.; Weickhardt, C.; Boesl, U.; Schlag, E. W. Int. J. Mass Spectrom. Ion Processes Vol. 44a (1989) pp. 1219-25 and Spengler, B.; Kirsch, D.; Kaufmann, R.; Jaeger, E. Rapid Commun. Mass Spectrom. 6 (1992) pp. 105-08). For product ion mass spectra, this approach has the same disadvantages as the time-slice method employed by Wiley and McLaren, in that it does not realize the full multiplex recording advantage of the time-of-flight mass spectrometer.
Although product ion mass spectra can be recorded in single TOF analyzers employing a reflectron, a number of investigators have described a variety of tandem configurations in which the first mass analyzer is utilized to select the precursor ion mass, while the second mass analyzer is used to record its product ion mass spectrum. Approaches using two linear TOF mass analyzers (i.e., without reflectrons) and reacceleration of the product ions have been described by Derrick (Jardine, D. R.; Morgan, J.; Alderdice, D. S.; Derrick, P. J.: Org. Mass Spectrom. Vol. 27 (1992) pp. 1077-83) and Cooks (Schey, K. L.; Cooks, R. G.; Grix, R; Wollnik, H., International Journal of Mass Spectrometry and Ion Processes Vol. 77 (1987) pp. 49-61).
A linear/reflectron (TOF/RTOF) configuration has also been reported by Cooks (Schey, K. L.; Cooks, R. G.; Kraft, A.; Grix, R.; Wollnik, H., International Journal of Mass Spectrometry and Ion Processes Vol. 94 (1989) pp. 1-14). Strobel and Russell (Strobel, F. H.; Solouki, T.; White, M. A.; Russell, D. H., J. Am. Soc. Mass Spectrom. Vol. 2 (1990) pp. 91-94); and (Strobel, F. H.; Preston, L. M.; Washburn, K. S.; Russell, D. H., Anal. Chem. Vol. 64 (1992) pp. 754-62) have recently described a hybrid instrument (EB/RTOF) using a double-focusing sector mass analyzer for mass selection and a reflectron TOF to record the product ions.
In addition, Cotter and Cornish (Cornish, T. J.; Cotter, R. J. Analytical Chemistry Vol. 65 (1993) pp. 1043-47, the entire content of which is hereby incorporated by reference) and (Cornish, T. J.; Cotter, R. J. Org. Mass Spectrom., the entire content of which is hereby incorporated by reference) have described a tandem (RTOF/RTOF) time-of-flight instrument using two reflecting time-of-flight mass analyzers. The first analyzer permits high resolution selection of the precursor ion by electronic gating prior to a collision cell, while the second mass analyzer is used to record the collision induced dissociation (CID) or product ion mass spectrum. In this instrument, both dual-stage and single-stage reflectrons have been used. However, both single and dual stage reflectrons currently suffer from the focusing limitations described above.
The tandem time-of-flight mass spectrometer has several clear advantages over the reflectron TOF analyzer for recording of product ion mass spectra. In many instances, these advantages resemble the advantages of a four sector (EBEB) instrument over the linked E/B scanning methods employed on two sector (EB) mass spectrometers.
That is, the tandem time-of-flight permits higher mass resolution selection of the precursor ion because electronic gating is accomplished as the ions are brought into time focus at the collision chamber. In contrast, ion mass gating in the first linear region (L1) of a reflectron TOF is carried out prior to focusing by the reflectron. Secondly, a tandem time-of-flight mass spectrometer incorporating two reflectrons can more clearly separate metastable processes from collision induced dissociation, since metastable ions that occur in the first field free region and traverse the first reflectron do not arrive at the ion mass gate at the same time.
In 1993 Enke and coworkers (Seterlin, M. A.; Vlasak, P. R.; McLane, R. D.; Enke, C. G., J. Am. Chem. Soc. 4 (1993) 751-754), also designed a tandem time-of-flight mass spectrometer, but used photodissociation to form the product ions. The focusing problem was adressed by decelerating the ions just prior to dissociation and reaccelerating the product ions into the second reflectron analyzer. However, this approach does not take full advantage of the full initial kinetic energy when collision induced dissociation is used. In a tandem instrument described by Vestal and co-workers (Medzihradsky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falik, A. M.; Juhasz, P.; Vestal, M. L.; Burlingham, A. L., Anal. Chem. 72 (2000) 552-558) and commercialized by applied biosystems of Framingham, Mass., ions are formed by Matrix Assisted Laser Desorption Ionization (MALDI) and focused by pulsed or delayed extraction to a focal point where the ions are mass selected by a timed ion gate. The ions then pass through a collision cell where they are dissociated. The product ions continue to have the same velocities as their mass selected precursors, so that they all enter a second “source” at the same time. They are then accelerated into a reflectron mass analyzer by pulsed extraction. In order to accommodate the limited bandwidth of the reflectron, the kinetic energy of the precursor ions (and hence the collision energy in the laboratory frame) is kept 1 to 2 keV, with pulsed extraction in the second source providing an additional 18 keV to the product ions. In this way, ions enter the reflectron with a range of energies for 18 to 20 keV. In an instrument designed at BRUKER DALTONICS from Bellerica, Mass., initial kinetic energies (and laboratory collision energies) are also set at few keV, with the additional acceleration of the product ions provided by raising the potential of a lift cell while the ions are in residence (Schnaible, V.; Wefing, S.; Resemann, A.; Suckau, D.; Bucker, A.; Wolf-Kummeth, Hoffman, D., Anal. Chem. 74 (2002) 4980-4988).